Volume 26, Issue 6, Pages 923-937 (December 2014) The Immunoreceptor TIGIT Regulates Antitumor and Antiviral CD8+ T Cell Effector Function  Robert J. Johnston, Laetitia Comps-Agrar, Jason Hackney, Xin Yu, Mahrukh Huseni, Yagai Yang, Summer Park, Vincent Javinal, Henry Chiu, Bryan Irving, Dan L. Eaton, Jane L. Grogan  Cancer Cell  Volume 26, Issue 6, Pages 923-937 (December 2014) DOI: 10.1016/j.ccell.2014.10.018 Copyright © 2014 Elsevier Inc. Terms and Conditions

Cancer Cell 2014 26, 923-937DOI: (10.1016/j.ccell.2014.10.018) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 TIGIT Expression Is Elevated in Human Cancer and Strongly Correlated with CD8 and PD-1 Gene expression analyses of human cancers were performed as described (see Experimental Procedures). Scatter plots show per-gene count data normalized by library size. Box and whisker plots show the variance-stabilized expression ratio of TIGIT and CD3ε. (A) Correlation of TIGIT and CD3ε RNA expression in LUSC (red) and normal lung (black). ρ = 0.86. Quantification of TIGIT/CD3ε expression ratios is also shown. LUSC ratio increase = 372%. ∗∗∗p = 1.46 × 10−46. (B) Correlation of TIGIT and CD3ε RNA expression in COAD (red) and normal colon (black). ρ = 0.83. Quantification of TIGIT/CD3ε expression ratios is also shown. COAD ratio increase = 116%. ∗∗∗p = 3.66 × 10−6. (C) Correlation of TIGIT and CD3ε RNA expression in UCEC (red) and normal uterine endometrium (black). ρ = 0.87. Quantification of TIGIT/CD3ε expression ratios is also shown. UCEC ratio increase = 419%. ∗∗∗p = 7.41 × 10−5. (D) Correlation of TIGIT and CD3ε RNA expression in BRCA (red) and normal breast (black). ρ = 0.82. Quantification of TIGIT/CD3ε expression ratios is also shown. BRCA ratio increase = 313%. ∗∗∗p = 4.6 × 10−44. (E) Correlation of TIGIT and CD3ε RNA expression in kidney renal clear cell carcinoma (red) and normal kidney (black). ρ = 0.94. Quantification of TIGIT/CD3ε expression ratios is also shown. (F) Correlation of TIGIT and CD8A (left) or TIGIT and CD4 (right) RNA expression in lung squamous cell carcinoma (red) and normal lung (black). ρ = 0.77 and 0.48, respectively. (G) Correlation of TIGIT and PD-1 (PDCD1) RNA expression in lung squamous cell carcinoma (red) and normal lung (black). ρ = 0.82. (H) Correlation of TIGIT and CD226 RNA expression in lung squamous cell carcinoma (red) and normal lung (black). ρ = 0.64. See also Figure S1 and Table S1. Cancer Cell 2014 26, 923-937DOI: (10.1016/j.ccell.2014.10.018) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 TIGIT and PD-1 Are Coordinately Expressed by Human and Murine Tumor-Infiltrating Lymphocytes (A–D) Analysis of lymphocytes from resected human NSCLC tumors, tumor-matched peripheral blood, and normal donor peripheral blood. Data are pooled from three independently acquired sets of samples. (A) Representative FACS plots of TIGIT expression by peripheral and tumor-infiltrating CD8+ T cells. Shown is the quantitation of TIGIT+ cells as a percentage of all CD8+ T cells. ∗p < 0.05. (B) Flow cytometry histogram representative of TIGIT expression by PD-1high (red) and PD-1low (blue) tumor-infiltrating CD8+ T cells. (C) Representative FACS plots of TIGIT expression by peripheral and tumor-infiltrating CD4+ T cells. Shown is the quantitation of TIGIT+ cells as a percentage of all CD4+ T cells. (D) Flow cytometry histogram representative of TIGIT expression by PD-1high (red) and PD-1low (blue) tumor-infiltrating CD4+ T cells. (E and F) BALB/c mice were inoculated with syngeneic CT26 colorectal carcinoma cells. Splenocytes and TILs were analyzed 14 days after inoculation when tumors had reached approximately 200 mm3 in size. Data are representative of more than three independent experiments (n = 5-6). Shown is a representative FACS plot of TIGIT expression by tumor-infiltrating (E) CD8+ T cells and (F) CD4+ T cells, with TIGIT+ cells boxed. Also shown is the quantitation of the frequency of TIGIT+ T cells as a percentage of all T cells. ∗p = 0.0134, ∗∗∗p < 0.0001. (G) Flow cytometry histogram representative of TIGIT expression by PD-1high and PD-1low tumor-infiltrating CD8+ T cells and by splenic CD8+ T cells. Quantitation of TIGIT MFI is also shown. ∗∗p = 0.0023. (H) Flow cytometry histogram representative of TIGIT expression by PD-1high and PD-1low tumor-infiltrating CD4+ T cells and by splenic CD4+ T cells. Quantitation of TIGIT MFI is also shown. ∗∗∗p = 0.0002. Error bars depict SEM. See also Figure S2. Cancer Cell 2014 26, 923-937DOI: (10.1016/j.ccell.2014.10.018) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 TIGIT and PD-L1 Blockades Synergistically Elicit Tumor Rejection (A–C) BALB/c mice were inoculated subcutaneously with CT26 colorectal carcinoma cells in their right thoracic flanks. When tumors reached approximately 200 mm3 in size, mice were treated with isotype control (black), anti-PD-L1 + control (red), anti-TIGIT + control (blue), or anti-PD-L1 + anti-TIGIT (purple) antibodies for 3 weeks. Data are representative of more than three independent experiments (A and B) or two independent experiments (C) [n = 10 (A and B) or 7–10 (C)]. (A) Median (left) and individual (right) CT26 tumor volumes over time. On day 12, ∗∗∗p < 0.001 between mice treated with anti-PD-L1 + anti-TIGIT and all other groups. (B) Mouse survival over time. (C) Approximately 60 days after initial inoculation, mice that received anti-TIGIT + anti-PD-L1 and reached CR, as well as naive control mice, were (re)inoculated with CT26 cells in their left thoracic flanks and inoculated with EMT6 breast carcinoma cells in their mammary fat pads. Median (left) and individual (right) tumor volumes for CT26 (squares) and EMT6 (triangles) in CR mice (purple and green) and naive mice (black and orange) are shown. See also Figure S3. Cancer Cell 2014 26, 923-937DOI: (10.1016/j.ccell.2014.10.018) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 TIGIT/PD-L1 Coblockade Efficacy Is Dependent on CD8+ T Cells (A and B) Wild-type BALB/c mice were inoculated with CT26 tumors. When tumors reached 100–150 mm3 in size, mice were temporarily depleted of CD8+ T cells and treated with anti-TIGIT + anti-PD-L1 or control antibodies as described in Figure 3. Data are representative of two independent experiments (n = 10). (A) Median (left) and individual (right) CT26 tumor volumes over time. (B) Quantitation of CT26 tumor volumes 17 days after the start of treatment. ∗∗∗p = 0.0004. (C) Wild-type BALB/c mice were inoculated with CT26 tumors and treated with anti-TIGIT + anti-PD-L1 and subsequently rechallenged with CT26 tumors (as described in Figure 3) with temporary depletion of CD8+ T cells at the time of rechallenge. Data are representative of two independent experiments (n = 5). Median (left) and individual (right) CT26 tumor volumes over time are shown. Error bars depict SEM. Cancer Cell 2014 26, 923-937DOI: (10.1016/j.ccell.2014.10.018) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 TIGIT Regulates Tumor-Infiltrating CD8+ T Cell Effector Function BALB/C mice were inoculated subcutaneously with CT26 colorectal carcinoma cells in their right thoracic flanks and treated with control, anti-PD-L1 + control, anti-TIGIT + control, or anti-PD-L1 + anti-TIGIT, as described in Figure 3. Tumor-draining lymph node (dLN)-resident and tumor-infiltrating T cells were analyzed by flow cytometry 7 days after the start of treatment. Data are representative of more than three independent experiments (n = 5). (A) Representative FACS plots of dLN-resident and tumor-infiltrating CD8+ T cells after stimulation ex vivo, with IFNγ+ cells boxed. (B) Quantitation of IFNγ-producing dLN-resident CD8+ and CD4+ T cells as percentages of total dLN-resident CD8+ and CD4+ T cells, respectively. IFNγ production by unstimulated (no stim.) T cells is also shown. ∗∗∗p < 0.001. (C) Quantitation of IFNγ-producing tumor-infiltrating CD8+ and CD4+ T cells as percentages of total tumor-infiltrating CD8+ and CD4+ T cells, respectively. IFNγ production by unstimulated T cells is also shown. ∗∗∗p = 0.0003. (D) Quantitation of IFNγ/TNFα dual-producing dLN-resident CD8+ and CD4+ T cells as percentages of total dLN resident CD8+ and CD4+ T cells, respectively. Dual cytokine production by unstimulated T cells is also shown. ∗∗p = 0.002, 0.003, and 0.001, respectively. (E) Quantitation of IFNγ/TNFα dual-producing tumor-infiltrating CD8+ and CD4+ T cells as percentages of total tumor-infiltrating CD8+ and CD4+ T cells, respectively. Dual cytokine production by unstimulated T cells is also shown. ∗∗∗p < 0.0001. Error bars depict SEM. See also Figure S4. Cancer Cell 2014 26, 923-937DOI: (10.1016/j.ccell.2014.10.018) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 TIGIT Enforces CD8+ T Cell Exhaustion during Chronic Viral Infection (A and B) C57BL6/J mice were infected with the Armstrong strain of LCMV, and splenocytes were analyzed 7 days after infection. Data are representative of two independent experiments (n = 5). (A) Flow cytometry histogram representative of TIGIT expression by naive (CD44lowCD62Lhigh) and effector (CD44highCD62Llow) CD4+ and CD8+ T cells. Quantitation of TIGIT+ cells as a percentage of naive and effector memory (Teff) CD4+ and CD8+ T cells is also shown. ∗∗∗p < 0.001. (B) Flow cytometry histogram representative of TIGIT expression by PD-1high and PD-1low effector CD8+ T cells. Quantitation of TIGIT MFI is also shown. ∗∗∗p < 0.001. (C) C57BL6/J mice were briefly depleted of CD4+ T cells and infected with the Clone 13 strain of LCMV. Splenocytes were analyzed 42 days after infection. Shown is a flow cytometry histogram representative of TIGIT expression by naive (CD44lowCD62Lhigh), central memory (CD44highCD62Lhigh), and effector memory (CD44highCD62Llow) CD8+ T cells. Quantitation of TIGIT MFI is also shown. ∗∗∗p < 0.001. Data are representative of two independent experiments (n = 5). (D–F) C57BL6/J mice were briefly depleted of CD4+ T cells and infected with the Clone 13 strain of LCMV. Mice were treated with isotype-matched control, anti-PD-L1 + control, anti-TIGIT + control, or anti-PD-L1 + anti-TIGIT antibodies starting 28 days after infection. Splenocytes and liver viral titers were analyzed 42 days after infection. Data are representative of two independent experiments (n = 10). (D) Quantitation of liver LCMV titers. ∗p = 0.0106, ∗∗p = 0.0047. (E) Representative FACS plots gated on CD8+ T cells, with activated cells (CD44highCD62Llow) boxed. Shown is the quantitation of activated cells as a percentage of total CD8+ T cells. ∗∗∗p < 0.0001. (F) Representative FACS plots gated on activated CD8+ T cells after stimulation in vitro, with IFNγ+ cells boxed. Quantitation of IFNγ-producing cells as a percentage of activated CD8+ T cells.∗p = 0.0352, ∗∗p = 0.0047. Error bars depict SEM. See also Figure S5. Cancer Cell 2014 26, 923-937DOI: (10.1016/j.ccell.2014.10.018) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 7 TIGIT Impairs CD226 Function by Directly Disrupting CD226 Homodimerization (A) CD8+ T cells were MACS-enriched from TIGITfl/fl CD4cre (CKO) and TIGITfl/fl CD4wt (WT) littermates and stimulated in the presence of anti-CD226 or isotype-matched control antibodies as indicated. H3-thymidine uptake is shown as a ratio relative to wild-type cells cultured without stimulation or treatment. ∗∗p = 0.0061, ∗∗∗p < 0.0001. Data are representative of two independent experiments (n = 5). (B) Wild-type C57BL6/J CD8+ T cells were MACS-enriched and stimulated in the presence of anti-TIGIT, anti-CD226, and/or isotype-matched control antibodies as indicated. H3-thymidine uptake is shown as a ratio relative to cells cultured without stimulation or treatment. ∗∗∗p < 0.001 in paired t tests. (C) Primary human CD8+ T cells were MACS-enriched from blood and stimulated with suboptimal levels of plate-bound anti-CD3 in the presence or absence of human recombinant PVR-Fc. Anti-TIGIT antibodies or isotype-matched control antibodies were added as indicated. Shown is the quantitation of 3H-thymidine uptake. ∗∗p = 0.0071 and 0.0014, respectively. (D) CHO cells were transiently transfected with increasing concentrations of acceptor (green “A”) and donor (gray “D”) FLAG-ST-CD226 as indicated. Shown is the quantification of FRET intensity relative to donor emission. Data are representative of three independent experiments (n = 3). (E and F) CHO cells were transiently transfected with human FLAG-ST-CD226 and with increasing concentrations of human HA-TIGIT as indicated. Data are representative of two or more independent experiments (n = 4). Data are normalized to the maximal signal. (E) Quantification of the CD226:CD226 FRET ratio. (F) Quantification of the TIGIT:CD226 FRET ratio. (G) Anti-FLAG (left) and anti-HA (right) immunoblots (IB) performed on either anti-FLAG or anti-HA immunoprecipitates (IP) prepared from COS-7 cells transfected with either an empty pRK vector or a combination of FLAG-CD226 and HA-TIGIT. Data are representative of two independent experiments. (H) CHO cells were transfected as in (F), and the TIGIT:CD226 FRET ratio was quantified after treatment with PBS (white) or anti-TIGIT (red). ∗∗∗p < 0.001. Data are representative of four independent experiments (n = 3). (I) Primary human T cells were MACS-enriched from blood and stimulated with anti-CD3 and anti-CD28. TIGIT+ and TIGIT− cells were sorted, rested, restimulated, and labeled for FRET with the indicated antibodies. Data are representative of two independent experiments. Shown is the quantification of FRET ratios. ∗∗∗p < 0.001. Error bars depict SEM. See also Figure S6. Cancer Cell 2014 26, 923-937DOI: (10.1016/j.ccell.2014.10.018) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 8 TIGIT Suppression of CD8+ T Cell Responses Is Dependent on CD226 (A–F) BALB/c mice were inoculated subcutaneously with CT26 colorectal carcinoma cells in their right thoracic flanks. When tumors reached approximately 200 mm3 in size, mice were treated with isotype control (black), anti-CD226 + control (orange), anti-PD-L1 + control (red), anti-TIGIT + anti-PD-L1 + control (purple), or anti-TIGIT + anti-PD-L1 + anti-CD226 (green) antibodies for 3 weeks. Data are representative of two independent experiments (n = 10 [A and B] or 5 [C–F]). (A) Median (left) and individual (right) CT26 tumor volumes over time. (B) Mouse survival over time. (C–F) After 7 days of treatment, tumor-infiltrating lymphocytes and tumor-draining lymph node-resident lymphocytes were assessed by flow cytometry. (C) Quantitation of IFNγ-producing CD8+ TILs as a percentage of total CD8+ TILs after stimulation in vitro. ∗∗p < 0.01. (D) Quantitation of IFNγ-producing cells as a percentage of tumor-draining lymph node CD8+ T cells after stimulation in vitro. ∗p < 0.05. (E) Quantitation of CD8+ TILs as a percentage of total TILs. ∗∗p < 0.01. (F) Quantitation of CD8+ T cells as a percentage of all tumor-draining lymph node-resident lymphocytes. Error bars depict SEM. See also Figure S7. Cancer Cell 2014 26, 923-937DOI: (10.1016/j.ccell.2014.10.018) Copyright © 2014 Elsevier Inc. Terms and Conditions